Hydrogels with intrinsic antibacterial activity prepared from naphthyl anthranilamide (NaA) capped peptide mimics

In this study, we prepared antibacterial hydrogels through the self-assembly of naphthyl anthranilamide (NaA) capped amino acid based cationic peptide mimics. These ultra-short cationic peptide mimics were rationally designed with NaA as a capping group, l-phenylalanine, a short aliphatic linker, and a cationic group. The synthesized peptide mimics efficiently formed hydrogels with minimum gel concentrations between 0.1 and 0.3%w/v. The resulting hydrogels exhibited desirable viscoelastic properties which can be tuned by varying the cationic group, electronegative substituent, or counter anion. Importantly, nanofibers from the NaA-capped cationic hydrogels were found to be the source of hydrogels’ potent bacteriacidal actvity against both Gram-positive and Gram-negative bacteria while remaining non-cytotoxic. These intrinsically antibacterial hydrogels are ideal candidates for further development in applications where bacterial contamination is problematic.

charges etc. This base scaffold provides a versatile platform for the systematic examination of the effects of different chemical moieties on intramolecular interactions, that induce self-assembly and gel formation, and on biological functions such as antimicrobial activity.
In this work, we present the hydrogel formation of a library of naphthyl anthranilamide (NaA) capped amino acid-based cationic peptide mimics. These ultra-short cationic peptide mimics composed of a NaA capping group, l-phenylalanine, a short aliphatic linker, and a cationic group (Fig. 1). These peptide mimics could be facilely synthesized on a gram scale via ring-opening reaction of isatoic anhydride in solution phase 25,26 , rather than using solid phase peptide synthesis (SPPS). As an effort to reduce the toxicity of the previously reported anthranilamide-based hydrogels, the design of current hydrogelators involves only one unit of phenylalanine. Apart from providing π-π stacking interaction, the aromatic side chain of phenylalanine also increases the overall hydrophobicity of the gelators.
Cationic charge was also introduced by incorporating either ammonium or guanidinium groups, which are attached through a short aliphatic linker, on the C-terminus of phenylalanine. The presence of cationic charge was also envisaged to aid the solubility of the resulting hydrogelators. Moreover, the cationic groups employed, have been reported to be responsible for antibacterial activity of AMPs and their mimics [27][28][29] . The antibacterial efficacy of the cationic hydrogels was evaluated in vitro against S. aureus and E. coli.
The structural, morphological, and mechanical properties of the cationic hydrogels were investigated using various techniques, namely 1 H NMR, circular dichroism (CD) spectroscopy, attenuated total reflectance infrared (ATR-FTIR), atomic force microscopy (AFM), and rheometer. The cytotoxicity was examined on HEK293T cells.

Result and discussion
NaA-capped ultra-short cationic hydrogelators design rationale. The NaA-capped cationic hydrogelators reported herein were systematically designed and synthesized with four modifications (Fig. 2), such as varying the linker length (Modification A), altering the cationic moiety (Modification B), introducing halogen (i.e. fluoro, chloro, and bromo) substituent (Modification C), and changing the counter anion (Modification D). The effect of each modification to the hydrogelation capability, mechanical properties, and antibacterial activity of the resulting hydrogels were evaluated.
As describe in Scheme S1, these peptide mimics were synthesized in excellent yields (60-78%).The NaAcapped cationic peptide mimics were synthesized on a gram scale via ring-opening reaction of isatoic anhydride derivatives using l-phenylalanine followed by amide coupling reaction to introduce the naphthyl moiety 25,26 . The cationic charge within these peptide mimics were introduced by either adding 1.5 equivalent of either glucono-∂-lactone (GdL), during the hydrogel formation, or treating the intermediate 3a with dilute acids (TFA or HCl).
Hydrogelation studies. The majority of the cationic peptide mimics formed hydrogels at physiological pH using a combination of physical and chemical triggers which could alter their thermodynamic equilibrium. Addition of GdL (1.5 equivalent) to compound 1a-8a (Fig. 3) and mild heating are required to generate the ammonium group, present in hydrogels 1-8. The presence of cationic group would lead to dissolution of these compounds in water (Scheme S2). Meanwhile, the cationic peptide mimics 9a and 10a (Fig. 3), bearing TFA − and Cl − as counter anion, were readily soluble in water after mild heating was applied to form hydrogels 9 and 10. All solutions turned clear upon heating, indicating complete disolution. Upon cooling to room temperature, the solutions became viscous within 3 h (Fig. S1).
These observations suggested that chemical modification of these peptide mimics affected their hydrogel formation capability. Although reported to increase the antibacterial activity of several peptide mimics 26,33 , the presence hydrophobic bromo substituent might interfere with the π-π stacking interaction of the aromatic group 5 . In addition, the choice of cationic group significantly influenced the balance between hydrophobicity www.nature.com/scientificreports/ and hydrophilicity of the overall molecules. Changing the cationic group from primary ammonium in hydrogel 2 (Log P = 2.7) to guanidinium in hydrogel 8 (Log P = 1.7) significantly increased the hydrophilicity of the peptide mimics, which may explain their unstable characteristics observed in vial inversion test. Due to their poor structural stability, further characterization of hydrogel 1, 5, and 8 was not carried out. The minimum gel concentration (MGC), the minimum concentration of peptide mimics required to form a self-supporting hydrogel, was qualitatively determined by varying the concentration of peptide mimics 2a-4a, 6a, 7a, 9a, and 10a in a vial inversion test [34][35][36] . These peptide mimics were found to form self-supporting hydrogels at low concentrations, with MGC ranging from 0.1 to 0.3%(w/v) (Table 1). Additionally, their MGC did not seem to be affected by chemical modifications.
Structural and morphological characterization of hydrogels. 1 H NMR analysis. In agreement with the visual observation obtained from vial inversion test, 1 H NMR spectra also indicated that self-assembly had occurred before the addition of NaCl. The 1 H NMR of peptide mimic 2a as a model compound, disolved in DMSO with GDL without heating (monomeric phase), exhibited well-resolved NMR features (Fig. 4, red). Conversely, at the same concentration (0.3%(w/v)) the 1 H NMR recorded from viscous solution in water showed notably broader peaks (Fig. 4, blue), indicating self-assembly had occured. Aggregates, including fibrils, frequently exhibit broadening of spectral features due to slower tumbling rates on the NMR time-scale 37 .
In addition, the NaA capping group was involved in the self-assembly of these cationic peptide mimics. Upon increasing the concentration from 0.1 to 0.3 %(w/v), an up-field chemical shift (Δδ = 0.06 ppm) of peaks that correspond to the aryl protons of the aromatic-capping group was observed. This indicates their participation in stacking interactions which drive the self-assembly (Fig. 4, green and blue) 25,38-40 .   www.nature.com/scientificreports/ Addition of NaCl was crucial to drive the equilibrium towards the self-assembled state, leading to selfsupporting hydrogel formation. The 1 H NMR spectrum of hydrogel 2 at 0.3%(w/v) with the addition of NaCl, showed extreme broadening and loss of 1 H NMR features that indicated complete transformation to their gel phase (Fig. 4, purple) 39 . At low ionic strength, the electrostatic repulsion between the charged molecules might create a barrier to limit self-assembly. Ionic solutes, from the salt, could screen the charges and mitigate the electrostatic repulsion to an extent that hydrogels could be formed 41 .
Secondary structure of hydrogel. The secondary structure of the self-assembled hydrogels was investigated by comparing the circular dichroism (CD) and ATR-FTIR spectra of the resulting hydrogels with the spectra of the corresponding peptide mimics. The result demonstrated that structural modification had only showed a subtle change to the CD ellipticity without affecting the over-all secondary structure of the resulting hydrogels (Fig. S2). The CD spectra obtained from hydrogels 2-4, 6, 7, 9, and 10 all exhibited the presence of a negative minimum at around 200 nm along with a weak positive maximum around 220 nm, suggesting the formation of a random/disordered coil 42,43 .
Morphology of 3-D network of hydrogels. The supramolecular morphologies of hydrogels made from NaAcapped peptide mimics were investigated using AFM. All the xerogels made from peptide mimics 2a-4a, 6a, 7a, www.nature.com/scientificreports/ 9a, and 10a exhibited nano fibrous network with a lot of junction zones which provided space responsible for immobilizing water molecules leading to hydrogel formation (Fig. 5). Chemical modifications resulted in variation of the fibers size (diameter) but did not dramatically alter the morphology of the resulting hydrogels (Table 2). Changes on the fiber diameter could affect the mechanical properties of the resulting hydrogel. For instance, thicker fibers, hence smaller pores, resulted in restricted molecule displacement which often correspond with formation of more robust hydrogel 45 .
Additionally, AFM was also employed to further confirmed that the self-assembly had occurred in the viscous solution without addition of salt. In agreement with the 1 H NMR and visual observation, fibers (74 ± 6 nm) lacking junction zones or bundling were observed from the viscous solution of peptide mimics 2a (Fig. 5h). This observation signified that the addition of salt to the viscous solution of peptide mimics 2a promotes fiber aggregation, which is pivotal in forming a self-supporting hydrogel.
Mechanical properties. For topical antibacterial application, AMPs have been mixed with hypromellose (a gel base), to obtain a more viscoelastic formulation 46,47 . The self-assembled properties of NaA-capped cationic peptide mimics allow for the formation of viscoelastic hydrogels without the presence of any additive. Frequency sweep test (FST) was performed using a rheometer to precisely examine the viscoelastic properties of the resulting hydrogels. From FST, all of the NaA-capped cationic peptide mimics showed viscoelastic properties consistent with the formation of stable hydrogels. As shown in Fig. S3, the G′ values of hydrogels made from peptide mimics 2a-4a, 6a, 7a, 9a, and 10a were at least a magnitude higher than their modulus loss (G″), which are indicative of elastic hydrogel characteristic rather than viscous materials 48,49 . In addition, their modulus storage  www.nature.com/scientificreports/ (G′) were also frequency independent, which confirmed the structural stability of these hydrogels, and were comparable with other reported peptide-based self-assembled therapeutic hydrogels [49][50][51][52] . Additionally, the mechanical rigidity of these hydrogels can be modulated by carefully altering the molecular design of the hydrogelators, particularly through variations of cationic group, substituent on the benzene ring of NaA capping group, and counter anion. G′ values often associated with mechanical rigidity of a hydrogel where higher G′ value ascribed to more robust hydrogels 48 . Changing the cationic group of the hydrogel from primary ammonium 2 to tertiary ammonium 3 and quaternary ammonium 4 significantly increase the G′ values from 0.83 to 8.63 kPa and 2.28 kPa, respectively ( Table 3). The presence of a methyl group in hydrogels 3 and 4 was thought to decrease their flexibility, which presumably leads to formation of more robust hydrogels 53 .
Moreover, the mechanical rigidity gradually decreased as larger electron withdrawing group (EWG) was introduced on the NaA-capping group. The presence of a relatively small EWG, i.e. fluoro in hydrogel 6, resulted in a decrease of G′ value from 8.63 to 1.20 kPa (Table 3). A further decrease in G′ value (0.53 kPa) was observed for hydrogel 7, bearing a chloro substituent. Nevertheless, both fluoro 6 and chloro 7 demonstrated prominent characteristics of stable hydrogels (Fig. S3). This observation suggested that the electronegative and steric effects of substituents can be used to fine-tune the mechanical properties of the resulting hydrogels 54 .
Furthermore, changing the anion from gluconate (hydrogel 3) to trifluoroacetate (hydrogel 9) resulted in a subtle change in the rigidity of the hydrogel. However, the hydrogel 10 (bearing a chloride anion) exhibited a notable decrease of G′ value, presumably due to the absence of H-bonding capability of the counter anion (Cl − ) 55 .
Antibacterial activity of the ultrashort cationic hydrogels. Staphylococcus aureus (S. aureus) is one of the major causative bacteria for skin and soft tissue infections 56,57 . Therefore, the NaA-capped cationic hydrogels were challenged against an inoculation of 3 × 10 4 CFUs/mL of S. aureus using a modification of a previously reported method 58 .
Hydrogel 2, bearing primary ammonium group, was found to be the most active and exhibited complete killing against S. aureus with a reduction of > 9.0 Log 10 CFUs/mL compared to the controls after 18 h growth (Fig. 6). This hydrogel demonstrated improved activity when compared to previously reported short peptidebased hydrogels in combating S. aureus [58][59][60][61] . Additionally, hydrogel 2 exhibited significant bacteria reduction (5.5 Log 10 ) even when it was tested against a higher inoculum of 10 8 CFUs/mL S. aureus colonies (Fig. S4).
The architecture of the cationic groups plays a significant role in determining the antibacterial activity in solution. For example, anthranilamide-derived peptide mimics bearing cationic charges have been reported to exhibit a range of antibacterial properties against Gram-positive (MIC 2.0-62.5 µM) and Gram negative (MIC 15.6-125 µM) bacteria 26 . Similarly, hydrogel having the tertiary ammonium 3a and quatenary ammonium 4a, showed 5.1 Log 10 and 4.1 Log 10 reductions, respectively. The presence of methyl substituent on the cationic moiety seems to reduce the antibacterial activity of the resulting hydrogels, presumably due to the decrease in membrane binding capability 62,63 .
In contrast to the finding by Kuppusamy et al. 26 , the presence of a halogen atom and variation of the counter anion did not dramatically alter the antibacterial properties of the parent hydrogel 3. The hydrogels bearing fluoro (6), chloro (7), TFA − (9), and Cl − (10) exhibited 3.1 Log 10 , 5.0 Log 10 , 4.9 Log 10 , and 3.0 Log 10 , respectively.
The rheological properties, specifically the storage modulus G′, was reported to play a significant role in determining the antibacterial properties of multi domain peptide (MDP) hydrogels by providing a mechanical support Table 3. Modulus storage (G′) and modulus loss (G″) of hydrogels made from NaA-capped cationic peptide mimics suggested formation of viscoelastic materials.  In contrast, no trend could be observed between the G′ value of the gels and their antibacterial activity in this study. This result suggests that the NaA-capped hydrogel inhibites bacteria growth in a different mechanism to MDP hydrogel.
To widen the scope of antibacterial study, the most active hydrogel against S. aureus made from primary ammonium 2a (hydrogel 2) was challenged against E. coli, a Gram-negative bacteria which is also associated with skin and soft tissue infection (SSTI) [65][66][67] . Hydrogel 2 showed notable antibacterial activity (6.4 Log 10 reduction) against E. coli (Fig. S5). The difference in antibacterial activity between Gram-positive and Gram-negative was presumably due to the presence of additional outer bilayer membrane consist of lipopolysaccharide and phospholipid in Gram-negative bacteria 68 .

Antibacterial activity of the fibers released from hydrogel 2. Mechanism of antibacterial actions
for most short peptide-based hydrogels is still not fully understood, however one plausible mechanism is the release of active substance from the parent hydrogel. To examine this hypothesis, hydrogel 2 were immersed in water over 10 days and the release of the substance was monitored using UV/Vis spectroscopy.
The UV/Vis spectra showed peaks which centered at 235 nm and 290 nm. This result suggested that the cationic peptide mimics was released from hydrogel 2 and could account for the antibacterial activity observed. After incubation at 37 °C for 24 h, 0.15 mg/mL (221 µM) was quantified to be released from primary ammonium hydrogel 2. Furthermore, a continuous release profile was observed over 9 days with ~ 14% of total peptide mimics was released at the end of experiment (Fig. 7).
Additionally, hydrogel 2 was subjected to a disk diffusion assay. A zone of inhibition with diameter of 2.3 cm was observed. This finding further demonstrates that an active agent was released from hydrogel 2.
AFM was performed on the supernatant solution to examine whether the supernatant solution was composed of monomers or intact nanofibers. Supramolecular nano fibers with diameter of 47 ± 4.9 nm were observed (Fig. 8a) despite the concentration of the peptide mimics at below their MGC (1 mg/mL) in the supernatant.
The antibacterial efficacy of the released nano fibers collected from supernatant solutions on day 1-9 were evaluated using a microdilution protocol 69 . The nano fibers were found to have a minimum inhibitory concentration (MIC) of 62 µM (Fig. 8c) against S. aureus. As seen in Fig. 7, the hydrogel released nanofibers above the active concentration within all 24 h periods across the 9 days. Supramolecular nano fibers have been reported to enhance the antibacterial activity, presumably due to higher local cationic densities on the fibrils 50 . Interestingly, the antibacterial activity of NaA-based cationic peptide mimics also relies on the self-assembled morphologies. At the same concentration (62 µM), cationic peptide mimics 2a (as a free compound) did not showed significant bacteria reductions (< 1Log 10 CFU/mL) (Fig. 8c, grey). AFM images obtained for short cationic peptide mimics 2a, which was prepared without forming the hydrogel, showed a mixture of fibers with spheroidal aggregates and lack of junction zones (Fig. 8b).
Cytotoxicity of hydrogel 2. Aside from exhibiting antibacterial properties, ideal candidates for antibacterial biomaterial need to exhibit low toxicity against mammalian cells. Therefore, the cytotoxicity of hydrogel 2 made from primary ammonium 2a, at various concentrations was examined against HEK293T cells.
More than 80% cells viability were observed indicating that hydrogel 2 was not toxic to normal cells (Fig. 9). The remarkable antibacterial activity and low toxicity of hydrogel 2 makes it a promising candidate for development as an antibacterial biomaterial.

Conclusions
A library of ultra-short NaA-capped hydrogelators having only one amino acid (l-phenylalanine) have been synthesised in excellent yields. Majority of the synthesised cationic peptide mimics efficiently formed hydrogels at low concentration under physiological pH. The resulting hydrogels adopted disordered coil as secondary structures and exhibited desirable mechanical properties, with rigidity modulated by either varying the cationic group, substituent on the anthranilamide-core, or counter anion. The peptide mimics are derived simply from readily available starting materials and the reactions are done in solution phase, which are more amenable to large-scale synthesis compared to current peptide based LMWG. We have also demonstrated the structural flexibility and versatile control over the many structural variations that can be constructed in NaA-capped gelators. This class of www.nature.com/scientificreports/ gelators represent a substantial departure from other LMWGs, enabling, for the first time, a systematic examination of the effects of different chemical moieties on intramolecular interactions which induce self-assembly and hydrogel formation, while also enabling function such as antibacterial activity. The NaA-capped cationic hydrogels, particularly primary ammonium 2, displayed promising antibacterial properties with significant bacteria reduction against S. aureus (> 9.0 Log 10 ) and E. coli (6.4 Log 10 ). Furthermore, the antibacterial activities of these hydrogels were attributed to the released of nanofibers. The most active hydrogel primary ammonium 2 demonstrated a continuous nanofiber released profile that was antibacterial over 9 days. Additionally, this hydrogel also exhibited low toxicity (> 80% viability) against HEK293T cells.
These intrinsically antibacterial and non-toxic hydrogels are ideal candidates for further development in applications where bacterial contamination is problematic.

Experimental
All chemicals and solvents used were purchased from Chemimpex, Combi Blocks, and Sigma Aldrich and were used directly without any further purification.
Synthesis. The Naphthyl anthranilamide-capped cationic peptide mimics 1a-10a were synthesized via ringopening reaction of isatoic anhydride derivatives as previously reported 25 . The scheme and synthetic procedures to obtain hydrogelators 1-10 along with their characterization data ( 1 H NMR, 13 C NMR, and HRMS) are given in the supplementary information.
Preparation of hydrogels. Respective amount of hydrogelator 1-10 was added to each vial with internal diameter (i.d.) of 10 mm containing Mili-Q water. For, hydrogelators 1-8, GdL (1.5 equivalents) was subsequently added. The resulting suspensions of 1-10 were slowly heated until all of the solid completely dissolved. Afterwards, NaCl (5 equivalents) was added to each vial to make up total volume of 1 mL, followed by gentle   Circular dichroism spectroscopy. Hydrogels 2-4, 6, 7, 9, and 10 were prepared at 1%w/v and were diluted 8 times in Mili-Q water before being transferred into a 0.2 mm path length cuvette. The spectra were collected using ChirascanPlus CD spectrometer (Applied Photophysic UK) scanning wavelengths of 180-500 nm with a bandwidth of 1 nm, 0.6 s per point, and step of 1 nm. Each experiment was performed in triplicate and the results were averaged into a single plot value.
Attenuated total reflectance infrared spectroscopy. The ATR-FTIR spectra were obtained using a Spectrum 100 FTIR spectrometer (PerkinElmer, USA) fitted with a 1 mm diamond-ZnSe crystal. Xerogels were made in situ by applying nitrogen flow to one drop of the pre-formed hydrogels (2%w/v) which was place on the ATR-crystal. The spectra of xerogels 2-4, 6, 7, 9, and 10 were recorded from 4000 to 650 cm −1 with a 4 cm −1 resolution and 4 scans. The spectra of D 2 O gels were measured by applying two drops of pre-formed gels at 2%w/v on the ATR crystal which was then recorded from 4000 to 650 cm −1 with a 4 cm −1 resolution and 4 scans.
Atomic force microscopy (AFM). Hydrogels 2-4, 6, 7, 9, and 10 were prepared from the corresponding cationic peptide mimics at their MGC, 2 × below, and 4 × below MGC in glass vials. Prior to gelation, one drop of these peptide mimics solutions was casted onto a mica substrate. Using a glass slide, each droplet was carefully spread and was left to dry overnight before imaging. Imaging was performed using a Bruker Multimode 8 Atomic Force Microscope in Scanasyst Air (PeakForce Tappings) mode, which is based on tapping mode AFM. To prevent damage of soft samples, the imaging parameters were constantly optimized through the force curves that were collected. Bruker Scanasyst-Air probes were used, with a spring constant of 0.4-0.8 N m −1 and a tip radius of 2 nm.
To gain some insight on the mechanism of antibacterial action, the released solutions from hydrogel 2 was directly casted on to a mica substrate. In addition, respective amount peptide mimics 2a and GdL were dissolved in DMSO-Water (5%: 95%) which then diluted to obtained concentration of 221 µM before casted on a mica substrate. After gently spread using a glass slide and left to dried overnight, these samples were imaged using a similar manner as describe above.
Rheology measurement. The mechanical properties of NaA-capped cationic hydrogels were assessed using an Anton Paar MCR 302 rheometer with a 25 mm stainless parallel plate configuration.
Initially, 1 mL of hydrogels were prepared from peptide mimics 2a-4a, 6a, 7a, 9a, and 10a in glass vials at concentration of 1%w/v. These vials were warmed using a heat gun to transform the hydrogel to the solution phase. Subsequently, 560 µL of the resulting solutions were cast onto the rheometer plate. The other plate was lowered to the measuring position (1 nm) and the hydrogel was allowed to stand for 3 h for the gel to form. Initially strain sweep test (SST) was performed at a frequency of 1 Hz using 0.1% strain to 100% strain to identified the linear viscoelastic region of each hydrogel. Subsequently, frequency sweep test (FST) was conducted at fixed strain of 0.1%, which is within the LVER of all hydrogels, and frequency ranging from 10 to 0.01 Hz. The rheology data were shown as the average of three repeats for each data point.
In vitro released of fibers from hydrogel 2. Hydrogel 2 was made at 1%w/v with final volume of 1 mL using the same method as described above. The resulting hydrogel was left to stand overnight at room temperature. PBS (1 mL) was added gently from the side wall to avoid physical fractures. Subsequently, the vial was incubated at 37 °C, where 1 mL of PBS was sampled at each time point and replaced with fresh PBS. The samples were subjected to 10 × dilution before quantified using UV-Vis spectroscopy. This experiment was performed in triplicate.
Antibacterial assay. A single colony of S. aureus 38 or E. coli K12 was grown overnight in Laura Brentani (LB) broth at 37 °C. The resulting suspension were centrifuged and re-suspended in the same volume of LB twice. The optical density (OD) of the resulting bacteria culture was adjusted to 0.1 at 600 nm in LB (10 8 CFUs/ mL) which further adjusted to 3 × 10 4 CFUs/mL. Bacteria solutions (1 mL), which contain either 10 8 CFUs/mL or 3 × 10 4 CFUs/mL, was gently added to hydrogel 2 at 1%w/v with total volume of 1 mL. After being incubated at 37 °C for 18 h, 100 µL of bacteria solution in each vial was taken and subjected to serial dilution using phosphate buffer solution (PBS). 20 µL of each dilution were carefully transferred into nutrient agar plates and incubated for another 18 h. The following day, bacterial growth inhibition was quantified using the viable count method. The experiment was performed twice in triplicate and multiple sample comparison was performed using oneway ANOVA. www.nature.com/scientificreports/ To further investigate their antibacterial activity, the released fibers from hydrogel 2 were tested against S. aureus 38. The solution taken from day 1 (contain ~ 221 uM) was adjusted to obtained final concentrations of 125, 62.5, 31.25, and 18 µM. On the other hand, monomers solution of cationic peptide mimics 2a, that has not formed hydrogels, was prepared by dissolving a known amount of peptide mimics 2a in DMSO to give a 20 mM stock solution which then diluted with LB to make up concentration of 125, 62.5, 31.25, and 18 µM. 100 µL of the above peptide mimics monomer and fiber solution was transferred to a 96 well plates.
Subsequently, 100 µL bacteria solution (10 6 CFUs/mL) was added to each well containing samples. A blank control was used: one containing 100 µL of bacteria solution and 100 µL of PBS. The plate was then incubated at 37 °C for 24 h. The following day, 20 µL solution from each well was subjected to a serial dilution then transferred onto agar plates followed by incubation at 37 °C for another 24 h. MIC value was determined as the lowest concentration that inhibited S. aureus growth on the agar plate 70 .
Cytotoxicity assays. Cytotoxicity measurements were performed in HEK293T cells using an alamarBlue colorimetric assay. Each experiment was repeated at least three times 25 . Cells were passaged using standard cell culture procedures. Cells were detached with trypsin and centrifuged (1000 rpm for 3 min). The supernatant was removed and the cells re-suspended in Dulbecco's Modified Eagle Medium (DMEM) at a concentration of 100,000 cells per mL. Cells were seeded at a concentration of 6,000 cells per well. For cytotoxicity measurements, 100 mL of hydrogel 2 at 0.3, 0.6, and 1.0%w/v were added in triplicate to a 96-well plate and allowed to set overnight. Surrounding wells were supplemented with water to ensure hydration of the gels. Gels were then incubated for 24 h with DMEM. Cells were seeded atop the hydrogels and incubated for 24 h. 10 mL of Alamar Blue was added to the wells, followed by further incubation for 4 h. Control wells included cell-free gels, no hydrogels and a negative control of 15% (v/v) DMSO. The absorbances at 570 nm and 596 nm were recorded using a BioRad Benchmark plate reader.

Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.